Phototropins Function in High-Intensity Blue Light-Induced Hypocotyl Phototropism in Arabidopsis by Altering Cytosolic Calcium

Phototropins (phot1 and phot2), the blue light receptors in plants, regulate hypocotyl phototropism in a fluence-dependent manner. Especially under high fluence rates of blue light (HBL), the redundant function mediated by both phot1 and phot2 drastically restricts the understanding of the roles of phot2. Here, systematic analysis of phototropin-related mutants and overexpression transgenic lines revealed that HBL specifically induced a transient increase in cytosolic Ca²⁺ concentration ([Ca2+]_cyt) in Arabidopsis (Arabidopsis thaliana) hypocotyls and that the increase in [Ca2+]_cyt was primarily attributed to phot2. Pharmacological and genetic experiments illustrated that HBL-induced Ca²⁺ increases were modulated differently by phot1 and phot2. Phot2 mediated the HBL-induced increase in [Ca2+]_cyt mainly by an inner store-dependent Ca²⁺-release pathway, not by activating plasma membrane Ca²⁺ channels. Further analysis showed that the increase in [Ca2+]_cyt was possibly responsible for HBL-induced hypocotyl phototropism. An inhibitor of auxin efflux carrier exhibited significant inhibitions of both phototropism and increases in [Ca2+]_cyt, which indicates that polar auxin transport is possibly involved in HBL-induced responses. Moreover, PHYTOCHROME KINASE SUBSTRATE1 (PKS1), the phototropin-related signaling element identified, interacted physically with phototropins, auxin efflux carrier PIN-FORMED1 and calcium-binding protein CALMODULIN4, in vitro and in vivo, respectively, and HBL-induced phototropism was impaired in pks multiple mutants, indicating the role of the PKS family in HBL-induced phototropism. Together, these results provide new insights into the functions of phototropins and highlight a potential integration point through which Ca²⁺ signaling-related HBL modulates hypocotyl phototropic responses.Blue light (BL) is a key factor controlling plant growth and morphogenesis. Recent genetics investigations using Arabidopsis (Arabidopsis thaliana) have revealed that the BL receptors phototropin1 (phot1) and phot2 mediate BL-induced plant movements such as phototropism, chloroplast relocation, stomatal opening, leaf flattening, and leaf positioning responses (Inoue et al., 2010). Most of these responses are mediated redundantly by both phot1 and phot2 (Kinoshita et al., 2001; Sakamoto and Briggs, 2002), but some responses are mediated by either phot1 or phot2 (Sakai et al., 2001; Suetsugu et al., 2005). In addition, several lines of evidence have indicated that phot2 might negatively regulate the phot1-mediated response (de Carbonnel et al., 2010) and vice versa (Harada et al., 2003, 2013).One of the numerous physiological processes controlled by BL is phototropism. Phototropism enables plants to bend toward incident light by perceiving the direction, wavelength, and intensity of incident light so that they are able to obtain optimum light. Genetic evidence has shown that both phot1 and phot2 redundantly function to regulate hypocotyl phototropism in a fluence-dependent manner (Sakai et al., 2001). Phot1 functions at both low (0.01–1 μmol m⁻² s⁻¹) and high (greater than 1 μmol m⁻² s⁻¹) fluence rates to mediate phototropic responses, but phot2 functions only at high fluence rates (Inada et al., 2004). The functional specification of phot1 and phot2 could be attributed to the differences in signal intermediates between phot1 and phot2 signaling pathways.Genetic analysis has illustrated that phot1 mediates hypocotyl phototropism via its downstream signal transducers NONPHOTOTROPIC HYPOCOTYL3 (NPH3; Motchoulski and Liscum, 1999), ROOT PHOTOTROPISM2 (RPT2; Sakai et al., 2000), and NONPHOTOTROPIC HYPOCOTYL4/AUXIN RESPONSE FACTOR7 (NPH4/ARF7; Harper et al., 2000), resulting in the asymmetric distribution of auxin and the induction of a phototropic response in higher plants. Recently, studies have demonstrated that PHYTOCHROME KINASE SUBSTRATE (PKS) proteins are required for hypocotyl phototropism and that PKS1 binds PHOT1 and NPH3 in vivo (Lariguet et al., 2006). In addition, ATP-BINDING CASSETTE B19 (ABCB19), a newly identified auxin transporter, has been reported to interact with phot1 to regulate the BL-dependent phototropism (Christie et al., 2011). However, little is known about phot2-mediated phototropism for functional specialization, especially under high fluence rates of blue light (HBL), although several lines of evidence have shown that phot2- and phot1-mediated signaling pathways share some intermediates in BL responses (Kimura and Kagawa, 2006; Christie, 2007). Previous researches have suggested that phot1 acts not only positively in the presence of RPT2 but also negatively in its absence during the phototropic response of hypocotyls at high fluence rates, suggesting that RPT2 modulates the function of phot1. However, RPT2 does not act in the phot2-mediated pathway (Inada et al., 2004). More recently, RCN1-1, the A1 subunit of Ser/Thr PROTEIN PHOSPHATASE2A (PP2A), has been identified to interact with phot2. While reduced PP2A activity enhances the activity of phot2, it does not enhance either phot1 dephosphorylation or the activity of phot1 in mediating phototropism (Tseng and Briggs, 2010).Besides these signal intermediates noted above, phototropins may also confer their effects through the change of ion homeostasis. Ca²⁺ is a case in point. Recent reports have demonstrated that phototropins mediate the mobilization of Ca²⁺ in response to BL and that phot1 and phot2 mediate Ca²⁺ increases with distinctive mechanism in leaf cells according to the changes of ambient light intensity (Harada and Shimazaki, 2007). Under low fluence rates of BL, phot1 solely mediated Ca²⁺ influx through the channels in the plasma membrane. Under HBL, the increase in cytosolic Ca²⁺ concentration ([Ca2+]_cyt) is primarily attributed to phot2-dependent Ca²⁺ release from the internal calcium stores as well as the plasma membrane Ca²⁺ channels. Interestingly, the inhibitory effects of phospholipase C (PLC) inhibitors on the BL-induced responses in the wild type are larger than those in the phot1 single mutant, which indicates that there are some functional interactions between phot1 and phot2 to induce the elevation of cytosolic Ca²⁺ (Harada et al., 2003).However, until now, the function of Ca²⁺ in the phototropin-mediated phototropism signaling process has remained largely unknown. Pharmacological experiments indicate that changes in [Ca2+]_cyt are required for the phot1-mediated inhibition of hypocotyl growth but not for phot1-mediated phototropism (Folta et al., 2003). Otherwise, electrophysiological studies indicate that phototropic bending involves changes in ion fluxes, including calcium (Babourina et al., 2004). Such divergent responses show that the link between phototropins and calcium has not been firmly established in the case of hypocotyl phototropism. In phototropism, the phot1-dependent relocalization of the auxin efflux carrier PIN-FORMED1 (PIN1) is required for auxin redistribution (Blakeslee et al., 2004), and the PINOID kinase influences the relocalization of PIN1 (Friml et al., 2004). Given that both the calmodulin-related protein TCH3 and the calcium-binding protein AtPBP1 can bind to the PINOID kinase (Benjamins et al., 2003), it would appear that the cross talk among phototropins, auxin, and calcium is an important event for phototropism.Here, we show that HBL induces increases in [Ca2+]_cyt, which are mostly attributed to the function of phot2, and that the increases in [Ca2+]_cyt are required for HBL-induced phototropism in Arabidopsis hypocotyls. We also demonstrate that PKS1 may integrate phototropins with auxin transport in phot2-dependent Ca²⁺ signaling, and we discuss the possible molecular link between phototropins and other potential signal elements in HBL-induced phototropism.     

Clathrin Light Chains Regulate Clathrin-Mediated Trafficking,Auxin Signaling,and Development in Arabidopsis

Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.     

The Chromatin-Remodeling Factor PICKLE Integrates Brassinosteroid and Gibberellin Signaling during Skotomorphogenic Growth in Arabidopsis

Plant cell elongation is controlled by endogenous hormones, including brassinosteroid (BR) and gibberellin (GA), and by environmental factors, such as light/darkness. The molecular mechanisms underlying the convergence of these signals that govern cell growth remain largely unknown. We previously showed that the chromatin-remodeling factor PICKLE/ENHANCED PHOTOMORPHOGENIC1 (PKL/EPP1) represses photomorphogenesis in Arabidopsis thaliana. Here, we demonstrated that PKL physically interacted with PHYTOCHROME-INTERACTING FACTOR3 (PIF3) and BRASSINAZOLE-RESISTANT1 (BZR1), key components of the light and BR signaling pathways, respectively. Also, this interaction promoted the association of PKL with cell elongation–related genes. We found that PKL, PIF3, and BZR1 coregulate skotomorphogenesis by repressing the trimethylation of histone H3 Lys-27 (H3K27me3) on target promoters. Moreover, DELLA proteins interacted with PKL and attenuated its binding ability. Strikingly, brassinolide and GA₃ inhibited H3K27me3 modification of histones associated with cell elongation–related loci in a BZR1- and DELLA-mediated manner, respectively. Our findings reveal that the PKL chromatin-remodeling factor acts as a critical node that integrates light/darkness, BR, and GA signals to epigenetically regulate plant growth and development. This work also provides a molecular framework by which hormone signals regulate histone modification in concert with light/dark environmental cues.     

Arabidopsis ERG28 Tethers the Sterol C4-Demethylation Complex to Prevent Accumulation of a Biosynthetic Intermediate That Interferes with Polar Auxin Transport

Sterols are vital for cellular functions and eukaryotic development because of their essential role as membrane constituents. Sterol biosynthetic intermediates (SBIs) represent a potential reservoir of signaling molecules in mammals and fungi, but little is known about their functions in plants. SBIs are derived from the sterol C4-demethylation enzyme complex that is tethered to the membrane by Ergosterol biosynthetic protein28 (ERG28). Here, using nonlethal loss-of-function strategies focused on Arabidopsis thaliana ERG28, we found that the previously undetected SBI 4-carboxy-4-methyl-24-methylenecycloartanol (CMMC) inhibits polar auxin transport (PAT), a key mechanism by which the phytohormone auxin regulates several aspects of plant growth, including development and responses to environmental factors. The induced accumulation of CMMC in Arabidopsis erg28 plants was associated with diagnostic hallmarks of altered PAT, including the differentiation of pin-like inflorescence, loss of apical dominance, leaf fusion, and reduced root growth. PAT inhibition by CMMC occurs in a brassinosteroid-independent manner. The data presented show that ERG28 is required for PAT in plants. Furthermore, it is accumulation of an atypical SBI that may act to negatively regulate PAT in plants. Hence, the sterol pathway offers further prospects for mining new target molecules that could regulate plant development.     

Osmotic Stress Responses and Plant Growth Controlled by Potassium Transporters in Arabidopsis

Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K⁺ uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K⁺ channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using ⁸⁶radioactive rubidium ion (⁸⁶Rb⁺) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.     

Stomatal Blue Light Response Is Present in Early Vascular Plants

Light is a major environmental factor required for stomatal opening. Blue light (BL) induces stomatal opening in higher plants as a signal under the photosynthetic active radiation. The stomatal BL response is not present in the fern species of Polypodiopsida. The acquisition of a stomatal BL response might provide competitive advantages in both the uptake of CO₂ and prevention of water loss with the ability to rapidly open and close stomata. We surveyed the stomatal opening in response to strong red light (RL) and weak BL under the RL with gas exchange technique in a diverse selection of plant species from euphyllophytes, including spermatophytes and monilophytes, to lycophytes. We showed the presence of RL-induced stomatal opening in most of these species and found that the BL responses operated in all euphyllophytes except Polypodiopsida. We also confirmed that the stomatal opening in lycophytes, the early vascular plants, is driven by plasma membrane proton-translocating adenosine triphosphatase and K⁺ accumulation in guard cells, which is the same mechanism operating in stomata of angiosperms. These results suggest that the early vascular plants respond to both RL and BL and actively regulate stomatal aperture. We also found three plant species that absolutely require BL for both stomatal opening and photosynthetic CO₂ fixation, including a gymnosperm, C. revoluta, and the ferns Equisetum hyemale and Psilotum nudum.Stomata regulate gas exchange between plants and the atmosphere (Zeiger, 1983; Assmann, 1993; Roelfsema and Hedrich, 2005; Shimazaki et al., 2007; Kim et al., 2010). Acquisition of stomata was a key step in the evolution of terrestrial plants by allowing uptake of CO₂ from the atmosphere and accelerating the provision of nutrients via the transpiration stream within the plant (Hetherington and Woodward, 2003; McAdam and Brodribb, 2013). Stomatal aperture is regulated by changes in the turgor of guard cells, which are induced by environmental factors and endogenous phytohormones. Light is a major factor in the promotion of stomatal opening, and the opening is mediated via two distinct light-regulated pathways that are known as photosynthesis- and blue light (BL)-dependent responses under photosynthetic active radiation (PAR; Vavasseur and Raghavendra, 2005; Shimazaki et al., 2007; Lawson et al., 2014).The photosynthesis-dependent stomatal opening is induced by a continuous high intensity of light, and the action spectrum for the stomatal opening resembles that of photosynthetic pigments in leaves (Willmer and Fricker, 1996). Both mesophyll and guard cells possess photosynthetically active chloroplasts, and their photosynthesis has been suggested to contribute to stomatal opening in leaves. The decrease in the concentration of intercellular CO₂ (Ci) caused by photosynthetic CO₂ fixation or some unidentified mediators and metabolites from mesophyll cells is supposed to elicit stomatal opening, although the exact nature of the events is unclear (Wong et al., 1979; Vavasseur and Raghavendra, 2005; Roelfsema et al., 2006; Mott et al., 2008; Lawson et al., 2014).BL-dependent stomatal opening requires a strong intensity of PAR as a background: weak BL solely scarcely elicits stomatal opening, but the same intensity of BL induces the fast and large stomatal opening in the presence of strong red light (RL; Ogawa et al., 1978; Shimazaki et al., 2007). Since such stomatal opening requires BL under the RL or PAR, we call the opening reaction a BL-dependent response of stomata. BL-dependent stomatal response takes place and proceeds in natural environments because the sunlight contains both BL and RL and facilitates photosynthetic CO₂ fixation (Assmann, 1988; Takemiya et al., 2013a). In this stomatal response, BL and PAR (BL, RL, and other wavelengths of light) seem to act as a signal and an energy source, respectively.The BL-dependent stomatal opening is initiated by the absorption of BL by phototropin1 and phototropin2 (Kinoshita et al., 2001), the plant-specific BL receptors, in guard cells followed by activation of the plasma membrane proton-translocating adenosine triphosphatase (H+-ATPase; Kinoshita and Shimazaki, 1999). Two newly identified proteins, protein phosphatase1 and blue light signaling1 (BLUS1), mediate the signaling between phototropins and H+-ATPase (Takemiya et al., 2006, 2013a, 2013b). The activated H+-ATPase evokes a plasma membrane hyperpolarization, which drives K⁺ uptake through the voltage-gated, inward-rectifying K⁺ channels (Assmann, 1993; Shimazaki et al., 2007; Kim et al., 2010; Kollist et al., 2014). The accumulation of K⁺ causes water uptake and increases turgor pressure of guard cells, and finally results in stomatal opening. The BL-dependent opening is enhanced by RL, and BL at low intensity is effective in the presence of RL (Ogawa et al., 1978; Iino et al., 1985; Shimazaki et al., 2007; Suetsugu et al., 2014). These stomatal responses by RL and BL are commonly observed in a number of seed plants so far examined.Fine control of stomatal aperture to various environmental factors has been characterized in many angiosperms. Although morphological and mechanical diversity of stomata is widely documented, little is known about the functional diversity (Willmer and Fricker, 1996; Hetherington and Woodward, 2003). Our previous study indicated that BL-dependent stomatal response is absent in the major fern species of Polypodiopsida, including Adiantum capillus-veneris, Pteris cretica, Asplenium scolopendrium, and Nephrolepis auriculata, but the stomata of these species open by PAR including RL (Doi et al., 2006). When the epidermal peels isolated from A. capillus-veneris are treated with photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1dimethylurea (Doi and Shimazaki, 2008), the response is completely inhibited, but the responses in the seed plants of Vicia faba and Commelina communis are relatively insensitive to 3-(3,4-dichlorophenyl)-1,1dimethylurea (Schwartz and Zeiger, 1984). These findings suggest that there is functional diversity in light-dependent stomatal response in different lineages of land plants. In accord with this notion, the different sensitivities of stomatal response to abscisic acid and CO₂ have been reported among the plant species of angiosperm, gymnosperm, ferns, and lycophytes (Mansfield and Willmer, 1969; Brodribb and McAdam, 2011), although the exact responsiveness to abscisic acid and CO₂ is still debated (Chater et al., 2011, 2013; Ruszala et al., 2011; McAdam and Brodribb, 2013).To address the origin and distribution of stomatal light responses, we investigated the presence of a stomatal response using a gas exchange method and various lineages of vascular plants, including euphyllophytes and lycophytes. Unexpectedly, all plant lineages except Polypodiopsida in monilophytes exhibited a stomatal response to BL in the presence of RL, suggesting that the response was present in the early evolutionary stage of vascular plants. We also report the stomatal opening in response to RL in these plant species.     

Phytochrome B Promotes Branching in Arabidopsis by Suppressing Auxin Signaling

Many plants respond to competition signals generated by neighbors by evoking the shade avoidance syndrome, including increased main stem elongation and reduced branching. Vegetation-induced reduction in the red light:far-red light ratio provides a competition signal sensed by phytochromes. Plants deficient in phytochrome B (phyB) exhibit a constitutive shade avoidance syndrome including reduced branching. Because auxin in the polar auxin transport stream (PATS) inhibits axillary bud outgrowth, its role in regulating the phyB branching phenotype was tested. Removing the main shoot PATS auxin source by decapitation or chemically inhibiting the PATS strongly stimulated branching in Arabidopsis (Arabidopsis thaliana) deficient in phyB, but had a modest effect in the wild type. Whereas indole-3-acetic acid (IAA) levels were elevated in young phyB seedlings, there was less IAA in mature stems compared with the wild type. A split plate assay of bud outgrowth kinetics indicated that low auxin levels inhibited phyB buds more than the wild type. Because the auxin response could be a result of either the auxin signaling status or the bud’s ability to export auxin into the main shoot PATS, both parameters were assessed. Main shoots of phyB had less absolute auxin transport capacity compared with the wild type, but equal or greater capacity when based on the relative amounts of native IAA in the stems. Thus, auxin transport capacity was unlikely to restrict branching. Both shoots of young phyB seedlings and mature stem segments showed elevated expression of auxin-responsive genes and expression was further increased by auxin treatment, suggesting that phyB suppresses auxin signaling to promote branching.The development of shoot branches is a multistep process with many potential points of regulation. After the formation of an axillary meristem in the leaf axil, an axillary bud may form through the generation of leaves and other tissues. The axillary bud may grow out to form a branch, or may remain dormant or semidormant for an indefinite period of time (Bennett and Leyser, 2006). In Arabidopsis (Arabidopsis thaliana), the position of the bud in the rosette is a strong determinant of its fate, with upper buds displaying greater outgrowth potential than lower buds. In fact, the varying potential of buds at different positions is maintained even in buds that are activated to form branches, with the upper buds growing out first and most robustly, and lower buds growing out after a time lag and with less vigor (Hempel and Feldman, 1994; Finlayson et al., 2010).The disparate fate of buds at different rosette positions is mediated, at least in part, by the process of correlative inhibition, whereby remote parts of the plant inhibit the outgrowth of the buds (Cline, 1997). Correlative inhibition is typically associated with the bud-inhibiting effects of auxin sourced in the shoot apex and transported basipetally in the polar auxin transport stream (PATS). Auxin in the PATS does not enter the bud and thus must act indirectly; however, the exact mechanism by which auxin inhibits bud outgrowth is not well understood, despite many years of intensive study (Waldie et al., 2010; Domagalska and Leyser, 2011). Evidence supports divergent models by which auxin may regulate branching. One model contends that the PATS modulates a bud outgrowth inhibiting second messenger (Brewer et al., 2009). Another model postulates a mechanism whereby competition between the main shoot and the axillary bud for auxin export in the PATS regulates bud activity (Bennett et al., 2006; Prusinkiewicz et al., 2009; Balla et al., 2011).In addition to intrinsic developmental programming, branching is also modulated by environmental signals, including competition signals generated by neighboring plants. The red light:far-red light ratio (R:FR) is an established competition signal that is modified (reduced) by neighboring plants and sensed by the phytochrome family of photoreceptors. A low R:FR evokes the shade avoidance syndrome with plants displaying, among other phenotypes, enhanced shoot elongation and reduced branching (Smith, 1995; Ballaré, 1999; Franklin and Whitelam, 2005; Casal, 2012). Phytochrome B (phyB) is the major sensor contributing to R:FR responses, and loss of phyB function results in a plant that displays a phenotype similar to constitutive shade avoidance. It should be noted that actual shade avoidance is mediated by additional phytochromes and that the complete absence of functional phyB in the loss-of-function mutant may also result in a phenotype that does not exactly mirror shade avoidance. Loss of phyB function leads to reduced branching and altered expression of genes associated with hormone pathways and bud development in the axillary buds (Kebrom et al., 2006; Finlayson et al., 2010; Kebrom et al., 2010; Su et al., 2011). In Arabidopsis, phyB deficiency differentially affects the outgrowth of buds from specific positions in the rosette and thus demonstrates an important function in the regulation of correlative inhibition (Finlayson et al., 2010; Su et al., 2011), a process known to be influenced by auxin. Many aspects of auxin signaling are dependent on AUXIN RESISTANT1 (AXR1), which participates in activating the Skip-Cullin-F-box auxin signaling module (del Pozo et al., 2002). Reduced auxin signaling resulting from AXR1 deficiency enabled phyB-deficient plants to branch profusely and reduced correlative inhibition, thus establishing auxin signaling downstream of phyB action (Finlayson et al., 2010). Although a link between auxin signaling and phyB regulation of branching was demonstrated, the details of the interaction were not discovered.The relationship between auxin and shade avoidance responses has been investigated in some detail. Auxin signaling was implicated in shade avoidance responses mediated by ARABIDOPSIS THALIANA HOMEOBOX PROTEIN2 in young Arabidopsis seedlings (Steindler et al., 1999). Rapid changes in leaf development resulting from canopy shade were also shown to involve TRANSPORT INHIBITOR RESPONSE1-dependent auxin signaling (Carabelli et al., 2007). A link between auxin abundance and the response to the R:FR was demonstrated in Arabidopsis deficient for the TRP AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) auxin biosynthetic enzyme (Tao et al., 2008). Young wild-type seedlings respond to a decreased R:FR by increasing indole-3-acetic acid (IAA) biosynthesis, accumulating IAA, increasing hypocotyl and petiole elongation, and increasing leaf elevation. However, these responses are reduced in plants deficient in TAA1. Subsequent studies confirmed the importance of auxin in responses to the R:FR (Pierik et al., 2009; Kozuka et al., 2010; Keller et al., 2011), and also identified the auxin transporter PIN-FORMED3 as essential for hypocotyl elongation responses in young seedlings (Keuskamp et al., 2010). In addition to the roles of auxin abundance and transport in the process, auxin sensitivity has also been implicated in shade avoidance. Several auxin signaling genes are direct targets of the phytochrome signaling component PHYTOCHROME INTERACTING FACTOR5 (PIF5), and these genes are misregulated in Arabidopsis deficient in either PHYTOCHROME INTERACTING FACTOR4 (PIF4) or PIF5 (Hornitschek et al., 2012; Sun et al., 2013). Auxin-responsive hypocotyl elongation and auxin-induced gene expression were also reduced in young seedlings of the pif4pif5 double mutant (Hornitschek et al., 2012), which show defects in shade avoidance responses (Lorrain et al., 2008).Although some aspects of the regulation of branching are now understood, there are still many gaps in our knowledge of the process, especially as related to the regulation of branching by light signals. Because auxin is known to play a major role in regulating branch development, and because recent studies have implicated auxin in general shade avoidance responses and specifically in the regulation of branching by phyB, the hypothesis that auxin homeostasis, transport, and/or signaling may contribute to the hypobranching phenotype of phyB-deficient plants was generated and tested, using a variety of physiological and molecular approaches.     

Constitutively Active Mitogen-Activated Protein Kinase Versions Reveal Functions of Arabidopsis MPK4 in Pathogen Defense Signaling

Plant mitogen-activated protein kinases (MAPKs) are involved in important processes, including stress signaling and development. In a functional yeast screen, we identified mutations that render Arabidopsis thaliana MAPKs constitutively active (CA). Importantly, CA-MAPKs maintain their specificity toward known activators and substrates. As a proof-of-concept, Arabidopsis MAPK4 (MPK4) function in plant immunity was investigated. In agreement with the phenotype of mpk4 mutants, CA-MPK4 plants were compromised in pathogen-induced salicylic acid accumulation and disease resistance. MPK4 activity was found to negatively regulate pathogen-associated molecular pattern-induced reactive oxygen species production but had no impact on callose deposition, indicating that CA-MPK4 allows discriminating between processes regulated by MPK4 activity from processes indirectly affected by mpk4 mutation. Finally, MPK4 activity was also found to compromise effector-triggered immunity conditioned by the Toll Interleukin-1 Receptor–nucleotide binding (NB)–Leu-rich repeat (LRR) receptors RPS4 and RPP4 but not by the coiled coil–NB-LRR receptors RPM1 and RPS2. Overall, these data reveal important insights on how MPK4 regulates plant defenses and establishes that CA-MAPKs offer a powerful tool to analyze the function of plant MAPK pathways.     

Non-Recognition-of-BTH4, an Arabidopsis Mediator Subunit Homolog,Is Necessary for Development and Response to Salicylic Acid

Salicylic acid (SA) signaling acts in defense and plant development. The only gene demonstrated to be required for the response to SA is Arabidopsis thaliana NON-EXPRESSER OF PATHOGENESIS-RELATED GENE 1 (NPR1), and npr1 mutants are insensitive to SA. By focusing on the effect of analogs of SA on plant development, we identified mutants in additional genes acting in the SA response. In this work, we describe a gene necessary for the SA Non-Recognition-of-BTH4 (NRB4). Three nrb4 alleles recovered from the screen cause phenotypes similar to the wild type in the tested conditions, except for SA-related phenotypes. Plants with NRB4 null alleles express profound insensitivity to SA, even more than npr1. NRB4 null mutants are also sterile and their growth is compromised. Plants carrying weaker nrb4 alleles are also insensitive to SA, with some quantitative differences in some phenotypes, like systemic acquired resistance or pathogen growth restriction. When weak alleles are used, NPR1 and NRB4 mutations produce an additive phenotype, but we did not find evidence of a genetic interaction in F1 nor biochemical interaction in yeast or in planta. NRB4 is predicted to be a subunit of Mediator, the ortholog of MED15 in Arabidopsis. Mechanistically, NRB4 functions downstream of NPR1 to regulate the SA response.     

Salt Stress Reduces Root Meristem Size by Nitric Oxide-Mediated Modulation of Auxin Accumulation and Signaling in Arabidopsis

The development of the plant root system is highly plastic, which allows the plant to adapt to various environmental stresses. Salt stress inhibits root elongation by reducing the size of the root meristem. However, the mechanism underlying this process remains unclear. In this study, we explored whether and how auxin and nitric oxide (NO) are involved in salt-mediated inhibition of root meristem growth in Arabidopsis (Arabidopsis thaliana) using physiological, pharmacological, and genetic approaches. We found that salt stress significantly reduced root meristem size by down-regulating the expression of PINFORMED (PIN) genes, thereby reducing auxin levels. In addition, salt stress promoted AUXIN RESISTANT3 (AXR3)/INDOLE-3-ACETIC ACID17 (IAA17) stabilization, which repressed auxin signaling during this process. Furthermore, salt stress stimulated NO accumulation, whereas blocking NO production with the inhibitor N^ω-nitro-l-arginine-methylester compromised the salt-mediated reduction of root meristem size, PIN down-regulation, and stabilization of AXR3/IAA17, indicating that NO is involved in salt-mediated inhibition of root meristem growth. Taken together, these findings suggest that salt stress inhibits root meristem growth by repressing PIN expression (thereby reducing auxin levels) and stabilizing IAA17 (thereby repressing auxin signaling) via increasing NO levels.Due to agricultural practices and climate change, soil salinity has become a serious factor limiting the productivity and quality of agricultural crops (Zhu, 2007). Worldwide, high salinity in the soil damages approximately 20% of total irrigated lands and takes 1.5 million ha out of production each year (Munns and Tester, 2008). In general, high salinity affects plant growth and development by reducing plant water potential, altering nutrient uptake, and increasing the accumulation of toxic ions (Hasegawa et al., 2000; Munns, 2002; Zhang and Shi, 2013). Together, these effects severely reduce plant growth and survival.Because the root is the first organ to sense high salinity, salt stress plays a direct, important role in modulating root system architecture (Wang et al., 2009). For instance, salt stress negatively regulates root hair formation and gravitropism (Sun et al., 2008; Wang et al., 2008). The role of salt in lateral root formation depends on the NaCl concentration. While high NaCl levels inhibit lateral root formation, lower NaCl levels stimulate lateral root formation in an auxin-dependent manner (Zolla et al., 2010; Ji et al., 2013). The root meristem plays an essential role in sustaining root growth (Perilli et al., 2012). Salt stress inhibits primary root elongation by suppressing root meristem activity (West et al., 2004). However, how this inhibition occurs remains largely unclear.Plant hormones are important intermediary signaling compounds that function downstream of environmental stimuli. Among plant hormones, indole-3-acetic acid (IAA) is thought to play a fundamental role in root system architecture by regulating cell division, expansion, and differentiation. In Arabidopsis (Arabidopsis thaliana) root tips, a distal auxin maximum is formed and maintained by polar auxin transport (PAT), which determines the orientation and extent of cell division in the root meristem as well as root pattern formation (Sabatini et al., 1999). PINFORMED (PIN) proteins, which are components of the auxin efflux machinery, regulate primary root elongation and root meristem size (Blilou et al., 2005; Dello Ioio et al., 2008; Yuan et al., 2013, 2014). The auxin signal transduction pathway is activated by direct binding of auxin to its receptor protein, TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN SIGNALING F-BOX (AFB), promoting the degradation of Aux/IAA proteins, releasing auxin response factors (ARFs), and activating the expression of auxin-responsive genes (Gray et al., 2001; Dharmasiri et al., 2005a; Kepinski and Leyser, 2005). Aux/IAA proteins are short-lived, nuclear-localized proteins that play key roles in auxin signal activation and root growth modulation (Rouse et al., 1998). Other hormones and stresses often regulate auxin signaling by affecting Aux/IAA protein stability (Lim and Kunkel, 2004; Nemhauser et al., 2004; Wang et al., 2007; Kushwah and Laxmi, 2014).Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants (He et al., 2004; Fernández-Marcos et al., 2011; Shi et al., 2012), including important roles in the regulation of root growth and development. NO functions downstream of auxin during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002). Exogenous auxin-induced NO biosynthesis is associated with nitrate reductase activity during lateral root formation, and NO is necessary for auxin-induced lateral root and root hair development (Pagnussat et al., 2002; Lombardo et al., 2006). Pharmacological and genetic analyses in Arabidopsis indicate that NO suppresses primary root growth and root meristem activity (Fernández-Marcos et al., 2011). Additionally, both exogenous application of the NO donor sodium nitroprusside (SNP) and overaccumulation of NO in the mutant chlorophyll a/b binding protein underexpressed1 (cue1)/nitric oxide overproducer1 (nox1) result in reduced PIN1 expression and auxin accumulation in root tips. The auxin receptors protein TIR1 is S-nitrosylated by NO, suggesting that this protein is a direct target of NO in the regulation of root development (Terrile et al., 2012).Because NO is a free radical, NO levels are dynamically regulated by endogenous and environmental cues. Many phytohormones, including abscisic acid, auxin, cytokinin, salicylic acid, jasmonic acid, and ethylene, induce NO biosynthesis (Zottini et al., 2007; Kolbert et al., 2008; Tun et al., 2008; García et al., 2011). In addition, many abiotic and biotic stresses or stimuli, such as cold, heat, salt, drought, heavy metals, and pathogens/elicitors, also stimulate NO biosynthesis (Zhao et al., 2009; Mandal et al., 2012). Salt stress stimulates NO and ONOO^– accumulation in roots (Corpas et al., 2009), but the contribution of NO to root meristem growth under salinity stress has yet to be examined in detail.In this study, we found that salt stress significantly down-regulated the expression of PIN genes and promoted AUXIN RESISTANT3 (AXR3)/IAA17 stabilization. Furthermore, salt stress stimulated NO accumulation, and pharmacological inhibition of NO biosynthesis compromised the salt-mediated reduction in root meristem size. Our results support a model in which salt stress reduces root meristem size by increasing NO accumulation, which represses PIN expression and stabilizes IAA17, thereby reducing auxin levels and repressing auxin signaling.     

Regulation of Auxin Homeostasis and Gradients in Arabidopsis Roots through the Formation of the Indole-3-Acetic Acid Catabolite 2-Oxindole-3-Acetic Acid

The native auxin, indole-3-acetic acid (IAA), is a major regulator of plant growth and development. Its nonuniform distribution between cells and tissues underlies the spatiotemporal coordination of many developmental events and responses to environmental stimuli. The regulation of auxin gradients and the formation of auxin maxima/minima most likely involve the regulation of both metabolic and transport processes. In this article, we have demonstrated that 2-oxindole-3-acetic acid (oxIAA) is a major primary IAA catabolite formed in Arabidopsis thaliana root tissues. OxIAA had little biological activity and was formed rapidly and irreversibly in response to increases in auxin levels. We further showed that there is cell type–specific regulation of oxIAA levels in the Arabidopsis root apex. We propose that oxIAA is an important element in the regulation of output from auxin gradients and, therefore, in the regulation of auxin homeostasis and response mechanisms.     

A Dominant Mutation in the Light-Oxygen and Voltage2 Domain Vicinity Impairs Phototropin1 Signaling in Tomato

In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A′α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A′α helix region in phototropic signaling of tomato.Being sessile in nature, plants have developed diverse sets of sensory mechanisms, integrating external cues such as light, water, and temperature to adapt their growth and development to the ambient environment. Plants have evolved a cohort of photoreceptors such as red/far-red light-sensing phytochromes (Chen and Chory, 2011), UV-A/blue light (BL)-sensing phototropins (Christie, 2007; Holland et al., 2009; Suetsugu and Wada, 2013), cryptochromes (Yu et al., 2010; Liu et al., 2011), Zeitlupe (ZTL)/Flavin-binding, Kelch repeat, F-box protein1/light-oxygen and voltage (LOV)-kelch protein2 members of the ZTL/ADAGIO putative family of photoreceptors (Suetsugu and Wada, 2013), and UV-B light-sensing UV-B resistance8 (Heijde and Ulm, 2012), enabling them to sense nearly the full range of the solar spectrum. One of the most visually obvious photoresponses of flowering plants involves the growth and orientation of organs toward or away from light, particularly during the early stages of growth and the establishment of seedlings (Iino, 1990) and during gap-filling situations in dense canopy conditions (Ballaré, 1999) for optimizing photosynthesis and interspecies/intraspecies competition. Several studies involving the relative effectiveness of different wavelengths of the solar spectrum as well as monitoring of lateral differences in light intensity revealed that the directional growth of plants is specifically mediated by the UV-A/blue region of the visible spectrum. Molecular genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants inhibited in hypocotyl curvature toward BL revealed that, among the UV-A light-/BL-specific photoreceptors, the phototropins perceive ambient light as a cue for directional growth (Liscum and Briggs, 1995; Kagawa et al., 2001).Phototropins have been identified in several plant species, ranging from the green alga Chlamydomonas reinhardtii to higher plants (Briggs et al., 2001). To date, two members of the phototropins have been reported from higher plants, phot1 and phot2, which share sequence homology (Sakai et al., 2001). Physiological analyses with Arabidopsis mutants lacking phot1 and phot2 have revealed that, in addition to regulating the hypocotyl curvature of seedlings toward BL (Huala et al., 1997; Christie et al., 1998), phototropins also regulate a diverse range of responses in flowering plants (Christie and Murphy, 2013; Hohm et al., 2013). These responses include chloroplast movements (Sakai et al., 2001), nuclear positioning (Iwabuchi et al., 2007), stomatal opening (Kinoshita et al., 2001), sun tracking (Inoue et al., 2008b), leaf expansion (Ohgishi et al., 2004), leaf movements (Inoue et al., 2005), leaf photomorphogenesis (Kozuka et al., 2011), leaf flattening (Sakamoto and Briggs, 2002), and the rapid inhibition of the growth of etiolated hypocotyls (Folta and Spalding, 2001).While both phot1 and phot2 overlap in function in regulating phototropism, chloroplast accumulation, leaf expansion, and stomatal opening, they also exhibit differential photosensitivity to BL, where phot1 is more sensitive to low-fluence BL than phot2. Both phot1 and phot2 redundantly regulate the chloroplast accumulation toward low-fluence BL, and phot2 exclusively regulates the chloroplast avoidance from high-fluence BL (Jarillo et al., 2001; Kagawa et al., 2001), while phot1 solely mediates the rapid inhibition of the elongation of etiolated hypocotyls (Folta and Spalding, 2001). Analysis of mutants downstream of blue light perception by phototropins revealed that the phototropin signaling branches out at an early step, and phot1 and phot2 trigger distinct photoresponses recruiting multiple signaling partners (Christie and Murphy, 2013; Hohm et al., 2013).Molecular characterizations have shown that phototropins are plasma membrane-associated Ser/Thr kinases containing a photosensory domain (Briggs and Christie, 2002) in the N-terminal region composed of two LOV domains (LOV1 and LOV2) and the kinase domain at the C-terminal end. The LOV1 and LOV2 domains bind the FMN as chromophore and are responsible for BL sensing by phototropin. Although phototropins characteristically possess two LOV domains, the photoregulation of phototropin activity is predominantly mediated by LOV2 (Christie, 2007). The exposure to BL also causes adduct formation between the FMN and the Cys residue in LOV domains and leads to the phosphorylation of phototropin, which is believed to be the primary step in the transmission of phototropic signals (Christie et al., 1998; Sakai et al., 2000). To decipher the functions of different domains of phototropins, many different substitution mutants of phototropins have been generated, which have enabled the elucidation of the functional significance of the different domains (Matsuoka and Tokutomi, 2005; Jones et al., 2007; Kong et al., 2007; Inoue et al., 2008a). Inoue et al. (2008a) showed that the BL-induced autophosphorylation of Ser-851 in the C-terminal kinase domain of phototropin is the primary step for initiating stomatal opening, phototropism, chloroplast accumulation, and leaf flattening. Mutational studies also revealed that the photosensory N-terminal domain of phototropin acts as a kinase inhibitor, where the LOV2 domain inhibits the activity of kinase domain by binding to it, and BL exposure is required for the dissociation of the LOV2 domain, enabling phosphorylation of the kinase domain (Matsuoka and Tokutomi, 2005; Jones et al., 2007).While our current understanding of phototropism has been greatly facilitated by the isolation of phototropins and their signaling mutants, the phot mutants identified to date are loss-of-function alleles. The lack of dominant-negative alleles or alleles with increased sensitivity to phototropic stimulus has hindered exploration into the roles of different domains of phot proteins in regulating phototropic signaling. In addition, the dearth of mutants defective in phototropin or phototropin-mediated responses has been a major bottleneck in furthering our understanding of the function of phototropins in crop species. Although phototropin homologs have been identified from a variety of crop species, including oat (Avena sativa; Zacherl et al., 1998), rice (Oryza sativa; Kanegae et al., 2000), and tomato (Solanum lycopersicum; Sharma et al., 2007; Sharma and Sharma, 2007), only the coleoptile phototropism1 mutant of rice has been isolated, which is defective in BL phototropism (Haga et al., 2005).Here, we report that in a mutant screen for nonphototropic seedlings under continuous BL, we recovered a strong dominant-negative mutation of phot1. The dominant-negative mutations are useful to elucidate redundant functions, as mutant protein in addition to suppressing its own functions can also suppress the function of its partners. The characterization of this new phot1 mutant revealed that the dominant activity is caused by the substitution of an Arg residue located in the A′α helix in the Hinge1 region between the LOV1 and LOV2 domains. Our study shows the functional importance of the A′α helix (Halavaty and Moffat, 2007) in regulating phot1-mediated signaling in tomato.